Not applicable.
The present invention relates generally to devices for the electrophoretic separation of molecules. Specifically, the invention relates to an apparatus in which protocols can be performed and which can then load samples directly into gels for electrophoresis of molecules, and to equipment adapted to accept this apparatus.
Electrophoresis is the process of separating molecules on the basis of the molecules"" migrations in an electric field. In an electric field, a molecule will migrate towards the pole that carries a charge opposite to the charge carried by the molecule. Electrophoresis in a polymeric gel, such as an acrylamide or an agarose gel, stabilizes the electrophoretic system against convective disturbances. Further, the polymeric gel provides a porous passageway through which the molecules must travel. Since larger molecules will travel more slowly through the passageways than smaller molecules, use of a polymeric gel permits the separation by molecular size. The molecules can then be visualized by treatment with appropriate stains. If greater resolution is desired, the molecules can be radioactively labeled and the gel then exposed to X-ray film which, when developed, will reveal the migration position of the molecules.
Both vertical and horizontal assemblies are used in gel electrophoresis. In a vertical apparatus, sample wells are formed in the same plane as the gel and are loaded vertically. Horizontal gels are formed horizontally in a tray, with sample wells positioned in a vertical plane. The samples then electrophorese horizontally through the gel. Vertical gels are used particularly in analysis of proteins and in DNA sequencing, while horizontal gels are used for PCR analysis, including medical diagnostics, forensic comparisons and identifications, and preparative scale DNA purification and analytical work, for restriction enzyme digests, and for Northern blots.
Conventionally, when a vertical gel is to be formed, two glass plates are placed on top of each other, separated by a spacer on each side, and are positioned in a gel forming stand. A plastic or rubber xe2x80x9ccomb,xe2x80x9d so called because it has a number of teeth, or prongs, extending downward from its lower edge, is placed in the space between the plates at the top of the assembly, and a liquid, typically of polyacrylamide, is carefully poured into the space between the plates up to the lower edge of the comb. In vertical gels intended for electrophoresis of proteins, the gel is poured to cover the prongs of the comb. When the comb is removed, these prongs typically leave rectangular impressions, or xe2x80x9cwells,xe2x80x9d in the edge of the gel. The gel is then covered with a standard xe2x80x9crunning bufferxe2x80x9d solution, and samples to be electrophoresed, which have usually been centrifuged, and are often also subjected to heating steps, are mixed with a dense xe2x80x9cloading solutionxe2x80x9d and then micropipetted, or drawn into and then injected from a fine gauge syringe, into the wells, a process known as xe2x80x9cloadingxe2x80x9d the wells. The loading solution usually also contains a dye so that the technician loading the wells can confirm visually the placement of the sample into the correct well.
In DNA sequencing, the procedure is slightly different. DNA sequencing typically employs a xe2x80x9cshark""s -toothxe2x80x9d comb, which when seen from the front has a flat top and a bottom with prongs shaped like upside-down triangles. The comb is placed in the top of the gel forming apparatus between the glass plates, with the flat side down, and the gel poured. When the gel has set, the comb is removed, to expose the flat surface of the gel and the comb is inverted so that the teeth point down. The comb is then lowered so that the teeth just touch the surface of the gel, with a triangular space open between the teeth, the top of which extends beyond the glass plates of the apparatus. The samples are then loaded through the open spaces between the teeth and run down the interior sides of the glass plates to sit on the flat surface of the gel, separated from each other by the teeth. Gels used for DNA sequencing, in particular, are typically quite thin (between 0.2 and 0.4 mm), requiring considerable care in handling and sample loading.
Horizontal gels are typically formed of liquid agarose poured into a tray, and a comb is positioned to hang into the agarose from above. Once the gel has set, the comb is removed, leaving wells formed by the prongs of the comb. Samples to be electrophoresed are again mixed with a dense xe2x80x9cloading solutionxe2x80x9d to reduce loss of the sample into the running buffer, and are loaded into the wells by a pipette or from a syringe. The electrophoresis of both vertical and horizontal gels is well known in the art and is set forth in, e.g., Sambrook et al., Molecular Cloning, A Laboratory Manual (2nd Ed. 1989) Cold Spring Harbor Laboratory, Cold Spring Harbor, N.Y.; and Harlow and Lane, Antibodies, A Laboratory Manual (1988), Cold Spring Harbor Laboratory, Cold Spring Harbor, N.Y.
Recently, the use of electrophoresis has undergone explosive growth. The Human Genome Project, which has undertaken to sequence the entire human genome, for example, currently consumes the efforts of a large number of laboratories, and programs are also underway to determine the complete DNA sequence of important pathogens and other organisms, including a number of plants. The use of DNA comparisons in forensic work and in medical diagnostics has also been increasing rapidly.
The high volume of DNA sequencing and other forms of electrophoresis has led to attempts to automate the process. One bottleneck in the efforts to automate electrophoresis in general, and DNA sequencing in particular, has been loading the samples in the wells. While automated equipment has been able to take over much of the repetitive work involved in preparing samples for electrophoresis, it has proven difficult to build equipment capable of loading individual wells, and especially hard to build equipment capable of loading a well in the space (only 0.2 to 0.4 mm) between the plates of a typical DNA sequencing gel. Thus, technicians load the bulk of these gels by hand.
Manual loading is generally undesirable, especially for high volume work such as sequencing. Not only does it slow the process, but it also introduces the chance for error in the order of the samples as the technician repetitively transfers sample after sample from tubes or microtiter plates to the wells. Further, manual loading increases the risk that the technician will be exposed to biohazards, or to radioactivity from radiolabeled samples. It also increases the risk of contamination of the samples due to handling errors such as a failure to change a pipette tip between samples.
Finally, the multiple transfers of samples between containers, such as centrifuge tubes and microtiter plates, and finally into the wells of a gel, are themselves undesirable. Macromolecules, which are the molecules to which electrophoresis is most often applied, are particularly susceptible to cleavage from shearing forces, which are created each time they are pipetted or transferred by syringe from one container to another. Therefore, the current multiple transfers of samples to and from microtiter plates and centrifuge tubes and finally into the wells of electrophoresis gels can alter or destroy the very molecules whose detection is sought.
Accordingly, a need exists in the art for a means of loading gels which is more amenable to automation. Further, a need exists for a means of loading gels which reduces the opportunity to alter the order of samples, reduces the exposure of lab workers to potential hazards, and reduces the number of times a particular sample has to be transferred between containers. The subject invention fills these and other needs.
The invention provides an xe2x80x9cin situxe2x80x9d loader for electrophoresis gels, comprising a body which includes at least one passage. The passage contains an inlet and an outlet, which are spaced along the passage in fluid communication. At least a part of the loader is of a dimension which permits insertion of that part of the loader into a gel holding portion of an electrophoresis apparatus.
Further, at least a portion of the bore or hollow of the passage can be enlarged, to define a reaction chamber. The passage can have a barrier located in the interior of the passage to prevent any macromolecules located in the passage, and particularly in the reaction chamber, from flowing to the outlet under normal gravity. The internal barrier will typically be located below (often immediately below) the reaction chamber.
The loader can contain one or more than one passages. The outlet or outlets of the passage can define a flat edge. The loader can be made of a material which has a low affinity for nucleic acids, for proteins, or for both.
The loader can further comprise a barrier inside or at the outlet, wherein the barrier is a permeable membrane. The permeable membrane can be covered, externally to the outlet, with a removable impermeable material. The removable impermeable material can be bonded, or thermally bonded, to the loader. The removable impermeable material can be a metal foil, a plastic film, or a metallized film. The outlet can also be covered by a removable metal, plastic, or rubber sleeve, or one made of another material inert to the reagents to be used which is capable of being fitted snugly to the loader and of withstanding the procedures to be employed in connection with the loader in question.
The barrier at the outlet can also be moveable or removable.
The loader can also have a section which is larger, typically in the thickness, than the portion of the loader intended for insertion into the gel electrophoresis or gel forming apparatus. The loader can transfer heat to and from the contents of the loader at a rate suitable for conducting the polymerase chain reaction. The passages of the loader can contain an electrode or electrical contact permitting direct access to electric current. A side of the loader other than the side through the molecules are intended to undergo electrophoresis may be made in whole or part of metal or of another material which conducts electricity, to permit current to reach a sample in the interior of a passage.
The loader can have outlets which are spaced more closely than are the corresponding inlets. Two or more passages of the loader can have not only individual inlets providing access to the respective individual passage, but also an additional, common inlet providing access to each of the passages in connection with the common inlet. One or more passages, or wells, of the loader can also be preloaded with one or more reagents.
The invention also provides a system comprising an xe2x80x9cin situxe2x80x9d loader for electrophoretic gels comprising a body with at least one passage defined therein, the passage including an inlet and an outlet, wherein the inlet and the outlet are spaced along the passage in fluid communication, and further wherein at least a part of the loader is of a dimension which permits insertion of that part of the loader into a gel holding portion of an electrophoresis apparatus, and a gel.
Further, the invention provides a method for separating macromolecules electrophoretically using a gel, wherein the macromolecules are loaded into a loader having an inlet in fluid communication with a hollow passage in fluid communication with an outlet, the macromolecules being drawn through said outlet into a gel upon application of an electric current.
The invention further provides a kit comprising xe2x80x9cin situxe2x80x9d loader for electrophoretic gels comprising a body with at least one passage defined therein, the passage including an inlet and an outlet, wherein the inlet and the outlet are spaced along the passage in fluid communication, and further wherein at least a part of the loader is of a dimension which permits insertion of that part of the loader into a gel holding portion of an electrophoresis apparatus, and a gel electrophoresis apparatus.
Further, the invention provides a centrifuge adapted to hold a loader as set forth above, and a system comprising such a centrifuge and such a loader. The centrifuge may have means to heat or to cool samples, or to do both.
Finally, the invention provides devices for performing PCR, adapted to hold a loader as described above.